­­Differential contribution of the subthreshold operating currents IT, Ih and IKir to the resonance of thalamocortical neurons

Membrane potential oscillations of thalamocortical (TC) neurons are believed to be involved in the generation and maintenance of brain rhythms that underlie global physiological and pathological brain states. These membrane potential oscillations depend on the synaptic interactions of TC neurons and their intrinsic electrical properties. These oscillations may be also shaped by increased output responses at a preferred frequency, known as intrinsic neuronal resonance. Here we combine electrophysiological recordings in mouse brain slices, modern pharmacological tools, dynamic clamp and computational modeling to study the ionic mechanisms that generate and modulate TC neuron resonance. We confirm findings of pioneering studies showing that most TC neurons display resonance which results from the interaction of the slow inactivation of the low threshold calcium current IT with the passive properties of the membrane. We also show that the hyperpolarization activated cationic current Ih is not involved in the generation of resonance, instead, it plays a minor role in the stabilization of TC neuron impedance magnitude due to its large contribution to the steady conductance. More importantly, we also demonstrate that TC neuron resonance is amplified by the inward rectifier potassium current IKir by a mechanism that hinges on its strong voltage dependent inward rectification (i.e. a negative slope conductance region) Accumulating evidence indicate that the ion channels that control the oscillatory behavior of TC neurons participate in pathophysiological processes. Results presented here points to IKir as a new potential target for therapeutic intervention.

Perinatal activation of ERα and ERβ but not GPER-1 masculinizes female rat caudate-putamen medium spiny neuron electrophysiological properties

Exposure to steroid sex hormones such as 17β-estradiol (estradiol) during early life potentially permanently masculinize neuron electrophysiological phenotype. In rodents, one crucial component of this developmental process occurs in males, with estradiol aromatized in the brain from testes-sourced testosterone. However, it is unknown whether most neuron electrophysiological phenotypes are altered by this early masculinization process, including medium spiny neurons (MSNs) of the rat caudate-putamen. MSNs are the predominant and primary output neurons of the caudate-putamen, and exhibit increased intrinsic excitability in females compared to males. Here we hypothesize that since perinatal estradiol exposure occurs in males, then a comparable exposure in females to estradiol or its receptor agonists would be sufficient to induce masculinization. To test this hypothesis, we injected perinatal female rats with estradiol or its receptor agonists and then later assessed MSN electrophysiology. Female and male rats on postnatal day 0 and 1 were systemically injected with either vehicle, estradiol, the ERα agonist PPT, the ERβ agonist DPN, or the GPER-1 agonist G1. On postnatal days 19±2 MSN electrophysiological properties were assessed using whole-cell patch clamp recordings. Estradiol exposure abolished increased intrinsic excitability in female compared to male MSNs. Exposure to either an ERα or ERβ agonist masculinized female MSN evoked action potential firing rate properties, while exposure to an ERβ agonist masculinized female MSN inward rectification properties. Exposure to ER agonists minimally impacted male MSN electrophysiological properties. These findings indicate that perinatal estradiol exposure masculinizes MSN electrophysiological phenotype via activation of ERα and ERβ.

Kir4.1/Kir5.1 channels possess strong intrinsic inward rectification determined by a voltage-dependent K+-flux gating mechanism

Inwardly rectifying potassium (Kir) channels are broadly expressed in both excitable and nonexcitable tissues, where they contribute to a wide variety of cellular functions. Numerous studies have established that rectification of Kir channels is not an inherent property of the channel protein itself, but rather reflects strong voltage dependence of channel block by intracellular cations, such as polyamines and Mg2+. Here, we identify a previously unknown mechanism of inward rectification in Kir4.1/Kir5.1 channels in the absence of these endogenous blockers. This novel intrinsic rectification originates from the voltage-dependent behavior of Kir4.1/Kir5.1, which is generated by the flux of potassium ions through the channel pore; the inward K+-flux induces the opening of the gate, whereas the outward flux is unable to maintain the gate open. This gating mechanism powered by the K+-flux is convergent with the gating of PIP2 because, at a saturating concentration, PIP2 greatly reduces the inward rectification. Our findings provide evidence of the coexistence of two rectification mechanisms in Kir4.1/Kir5.1 channels: the classical inward rectification induced by blocking cations and an intrinsic voltage-dependent mechanism generated by the K+-flux gating.

Intracellular NASPM allows an unambiguous functional measure of GluA2-lacking calcium-permeable AMPA receptor prevalence

Calcium-permeable AMPA-type glutamate receptors (CP-AMPARs) contribute to many forms of synaptic plasticity and pathology. They can be distinguished from GluA2-containing calcium-impermeable AMPARs by the inward rectification of their currents, which reflects voltage-dependent block by intracellular spermine. However, the efficacy of this weakly permeant blocker is differentially altered by the presence of AMPAR auxiliary subunits – including transmembrane AMPAR regulatory proteins, cornichons and GSG1L – that are widely expressed in neurons and glia. This complicates the interpretation of rectification as a measure of CP-AMPAR expression. Here we show that inclusion of the spider toxin analogue 1-naphthylacetyl spermine (NASPM) in the intracellular recording solution results in complete block of GluA1-mediated outward currents irrespective of the type of associated auxiliary subunit. In neurons from GluA2-knockout mice expressing only CP-AMPARs, intracellular NASPM, unlike spermine, blocks all outward synaptic current. Thus, our results identify an unambiguous functional measure, sensitive solely to changes in CP-AMPAR prevalence.

Optogenetic actuation in ChR2-transduced fibroblasts alter excitation-contraction coupling and mechano-electric feedback in coupled cardiomyocytes: a computational modeling study

<abstract> <p>With the help of the conventional electrical method and the growing optogenetic technology, cardiac fibroblasts (Fbs) have been verified to couple electrically with working myocytes and bring electrophysiological remodeling changes in them. The intrinsic properties of cardiac functional autoregulation represented by excitation-contraction coupling (ECC) and mechano-electric feedback (MEF) have also been extensively studied. However, the roles of optogenetic stimulation on the characteristics of ECC and MEF in cardiomyocytes (CMs) coupled with Fbs have been barely investigated. In this study, we proposed a combined model composed of three modules to explore these influences. Simulation results showed that (1) during ECC, an increased light duration (LD) strengthened the inflow of ChR2 current and prolonged action potential duration (APD), and extended durations of twitch and internal sarcomere deformation through the decreased dissociation of calcium with troponin C (CaTnC) complexes and the prolonged duration of Xb attachment-detachment; (2) during MEF, an increased LD was followed by a longer muscle twitch and deformation, and led to APD prolongation through the inward ChR2 current and its inward rectification kinetics, which far outweighed the effects of the delaying dissociation of CaTnC complexes and the prolonged reverse mode of Na⁺-Ca²⁺ exchange on AP shortening; (3) due to the ChR2 current's rectification feature, enhancing the light irradiance (LI) brought slight variations in peak or valley values of electrophysiological and mechanical parameters while did not change durations of AP and twitch and muscle deformation in both ECC and MEF. In conclusion, the inward ChR2 current and its inward rectification feature were found to affect significantly the durations of AP and twitch in both ECC and MEF. The roles of optogenetic actuation on both ECC and MEF should be considered in future cardiac computational optogenetics at the tissue and organ scale.</p> </abstract>

Screening Technologies for Inward Rectifier Potassium Channels: Discovery of New Blockers and Activators

K+ channels play a critical role in maintaining the normal electrical activity of excitable cells by setting the cell resting membrane potential and by determining the shape and duration of the action potential. In nonexcitable cells, K+ channels establish electrochemical gradients necessary for maintaining salt and volume homeostasis of body fluids. Inward rectifier K+ (Kir) channels typically conduct larger inward currents than outward currents, resulting in an inwardly rectifying current versus voltage relationship. This property of inward rectification results from the voltage-dependent block of the channels by intracellular polyvalent cations and makes these channels uniquely designed for maintaining the resting potential near the K+ equilibrium potential (EK). The Kir family of channels consist of seven subfamilies of channels (Kir1.x through Kir7.x) that include the classic inward rectifier (Kir2.x) channel, the G-protein-gated inward rectifier K+ (GIRK) (Kir3.x), and the adenosine triphosphate (ATP)-sensitive (KATP) (Kir 6.x) channels as well as the renal Kir1.1 (ROMK), Kir4.1, and Kir7.1 channels. These channels not only function to regulate electrical/electrolyte transport activity, but also serve as effector molecules for G-protein-coupled receptors (GPCRs) and as molecular sensors for cell metabolism. Of significance, Kir channels represent promising pharmacological targets for treating a number of clinical conditions, including cardiac arrhythmias, anxiety, chronic pain, and hypertension. This review provides a brief background on the structure, function, and pharmacology of Kir channels and then focuses on describing and evaluating current high-throughput screening (HTS) technologies, such as membrane potential-sensitive fluorescent dye assays, ion flux measurements, and automated patch clamp systems used for Kir channel drug discovery.

Computational Study to Identify the Effects of the KCNJ2 E299V Mutation in Cardiac Pumping Capacity

The KCNJ2 gene mutations induce short QT syndrome (SQT3) by directly increasing the IK1 current. There have been many studies on the electrophysiological effects of mutations such as the KCNJ2 D172N that cause the SQT3. However, the KCNJ2 E299V mutation is distinguished from other representative gene mutations that can induce the short QT syndrome (SQT3) in that it increased IK1 current by impairing the inward rectification of K+ channels. The studies of the electromechanical effects on myocardial cells and mechanisms of E299V mutations are limited. Therefore, we investigated the electrophysiological changes and the concomitant mechanical responses according to the expression levels of the KCNJ2 E299V mutation during sinus rhythm and ventricular fibrillation. We performed excitation-contraction coupling simulations using a human ventricular model with both electrophysiological and mechanical properties. In order to observe the electromechanical changes due to the expression of KCNJ2 E299V mutation, the simulations were performed under normal condition (WT), heterogeneous mutation condition (WT/E299V), and pure mutation condition (E299V). First, a single-cell simulation was performed in three types of ventricular cells (endocardial cell, midmyocardial cell, and epicardial cell) to confirm the electrophysiological changes and arrhythmogenesis caused by the KCNJ2 E299V mutation. In three-dimensional sinus rhythm simulations, we compared electrical changes and the corresponding changes in mechanical performance caused by the expression level of E299V mutation. Then, we observed the electromechanical properties of the E299V mutation during ventricular fibrillation using the three-dimensional reentry simulation. The KCNJ2 E299V mutation accelerated the opening of the IK1 channel and increased IK1 current, resulting in a decrease in action potential duration. Accordingly, the QT interval was reduced by 48% and 60% compared to the WT condition, for the WT/E299V and E299V conditions, respectively. During sustained reentry, the wavelength was reduced due to the KCNJ2 E299V mutation. Furthermore, there was almost no ventricular contraction in both WT/E299V and E299V conditions. We concluded that in both sinus rhythm and fibrillation, the KCNJ2 E299V mutation results in very low contractility regardless of the expression level of mutation and increases the risk of cardiac arrest and cardiac death.

Expression and Ion Transport Activity of Rice OsHKT1;1 Variants

OsHKT1;1 in rice, belongs to the high-affinity K+ Transporter family, has been found to be involved in salt tolerance. OsHKT1;1 in japonica rice (Nipponbare) produces mRNA variants, but their functions remain elusive. In salt tolerant rice, Pokkali, eight OsHKT1;1 variants (V1-V8) were identified in addition to the full-length OsHKT1;1 (FL) cDNA. Absolute quantification by qPCR revealed that accumulation of OsHKT1;1-FL mRNA is minor in contrast to that of OsHKT1;1-V1, -V2, -V4, and -V7 mRNAs, all of which are predominant in shoots, while only V1 and V7 mRNAs are predominant in roots. Two electrode voltage clamp (TEVC) experiments using Xenopus laevis oocytes revealed that oocytes-expressing OsHKT1;1-FL from Pokkali exhibited inward-rectified currents in the presence of 96 mM Na+ as reported previously. Further TEVC analyses indicated that six of eight OsHKT1;1 variants elicited currents in a Na+ or a K+ bath solution. OsHKT1;1-V6 exhibited a similar inward rectification to the FL protein. Contrastingly, however, the rests mediated bidirectional currents in both Na+ and K+ bath solutions. These data suggest possibilities that novel mechanisms regulating the transport activity of OsHKT1;1 might exist, and that OsHKT1;1 variants might also carry out distinct physiological roles either independently or in combination with OsHKT1;1-FL.